Automated determination of arteriovenous ratio in images of blood vessels

ABSTRACT

The methods and systems provided can automatically determine an Arteriolar-to-Venular diameter Ratio, AVR, in blood vessels, such as retinal blood vessels and other blood vessels in vertebrates. The AVR is an important predictor of increases in the risk for stroke, cerebral atrophy, cognitive decline, and myocardial infarct.

CROSS REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation of U.S. Non-Provisional applicationSer. No. 13/355,386, filed Jan. 20, 2012, which claims priority to U.S.Provisional Application No. 61/434,551, filed Jan. 20, 2011, hereinincorporated by reference in their entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under EY017066 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SUMMARY

The methods and systems provided can automatically determine anArteriolar-to-Venular diameter Ratio, AVR, in blood vessels, such asretinal blood vessels and other blood vessels in vertebrates. The AVR isan important predictor of increases in the risk for stroke, cerebralatrophy, cognitive decline, and myocardial infarct. It can thus be usedto perform risk analysis of (a) cardiovascular events in patientsalready undergoing fundus imaging for other reasons, such as diabeticretinopathy screening, (b) risk analysis for patients undergoing fundusimaging for specifically that reason, or (c) risk analysis in othersituations.

The methods and systems provided can predict cardiovascular events. Indifferent embodiments, the methods and systems provided can beapplicable in many areas including Medicine, Neurology, Primary Care,Ophthalmology, as well as other applications to determine properties ofblood vessels in other parts of the body that are suitable formulti-wavelength imaging, such as the iris, the skin, the eardrum, aswell as other organs in albino animals.

Until now, the AVR has always been determined manually from retinalcolor fundus images, which is a time consuming process requiring anexpert. Though clinicians look at the retina, they can only performgross estimates for substantially abnormal a/v ratios, and are incapableof determining numeric ratios.

A decreased ratio of the width of retinal arteries to veins(Arteriolar-to-Venular diameter Ratio, AVR), can be predictive ofmedical conditions such as cerebral atrophy, stroke and othercardiovascular events. Tortuous and dilated arteries and veins, as wellas decreased AVR can also be a marker for Plus disease in retinopathy ofprematurity. In an aspect, the methods and systems provided can estimatethe AVR in retinal color images by detecting the location of the opticdisc, determining an appropriate region of interest (ROI), classifyingvessels as arteries or veins, estimating the widths and calculating theAVR. In a further aspect, after vessel segmentation and vessel widthdetermination, the optic disc is located and the system can eliminateall vessels outside the AVR measurement ROI. A skeletonization operationcan be applied to the remaining vessels after which vessel crossings andbifurcation points can be removed, leaving a set of vessel segments thatare vessel centerline pixels. Features can be extracted from eachcenterline pixel in order to assign these a soft label indicating thelikelihood the pixel is part of a vein. As all centerline pixels in aconnected vessel segment should be the same type, the median soft labelcan be assigned to each centerline pixel in the segment. Artery/veinpairs can be matched using an iterative algorithm, and the widths of thevessels used to calculate the AVR.

The automated method and systems provided for determination of the AVRcan have substantial impact on clinical practice, and an improvedassessment for patients at risk for cardiovascular and brain disease.

Additional advantages will be set forth in part in the description whichfollows or may be learned by practice. The advantages will be realizedand attained by means of the elements and combinations particularlypointed out in the appended claims. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments and together with thedescription, serve to explain the principles of the methods and systems:

FIG. 1 shows an image overlaid with an automatically determined ROIregion;

FIG. 2A shows a color fundus image;

FIG. 2B shows a vessel likelihood map;

FIG. 2C shows a splat map, the borders of the individual splats areshown;

FIG. 2D shows a processed vessel likelihood map;

FIG. 2E shows vessel centerlines where each centerline pixel has beenassigned the likelihood it is inside a vein (dilated for display);

FIG. 2F shows final width measures overlaid on the original image shownin FIG. 2A;

FIG. 3A shows a retinal image;

FIG. 3B shows an artery vein centerline classification result overlaidon the retinal image shown in FIG. 3A;

FIG. 4 shows a detail of a fundus photograph showing the measurementdiameters and the vessel crossings where width measurements areobtained;

FIG. 5A shows a color fundus image;

FIG. 5B shows the corresponding vesselness image of the color fundusimage shown in FIG. 5A;

FIG. 6A shows a small segment of a vessel extracted from FIG. 5A or FIG.5B;

FIG. 6B shows a vessel probability image of FIG. 6A;

FIG. 6C shows a vessel centerline image of FIG. 6A;

FIG. 6D shows vessel growing directions;

FIG. 6E shows a graph depiction of vessel growing directions;

FIG. 7A shows an enlarged part of a channel image;

FIG. 7B shows an orientation sensitive 1D derivative of Gaussian result;

FIG. 8 shows an exemplary flowchart illustrating steps of a method;

FIG. 9 shows a curve from a ROC analysis;

FIG. 10A provides Bland-Altman plots or the agreement between anautomatic system and a reference standard;

FIG. 10B provides Bland-Altman plots or the agreement between a secondobserver and the reference standard;

FIG. 11A shows an image from the CLRIS database;

FIG. 11B shows an enlarged part of the image shown in FIG. 11A;

FIG. 12 shows a correlation of predicted vessel width and a mean of theobservers' measurement for HRIS;

FIG. 13 illustrates a relationship between vessel width and distance tooptic disc;

FIG. 14A shows a test vessel segment from HRIS

FIG. 14B shows a test vessel segment from KPIS;

FIG. 14C shows the vessel width measurement result of FIG. 14A;

FIG. 14D shows the vessel width measurement result of FIG. 14B;

FIG. 14E shows the cross-sectional view of vessel intensity with regardto the distance to the centerline for FIG. 14A;

FIG. 14F shows a cross-sectional view of vessel intensity with regard toa distance to a centerline of the test vessel segment shown in FIG. 14B;and

FIG. 15 is an exemplary operating environment.

DETAILED DESCRIPTION

Before the present methods and systems are disclosed and described, itis to be understood that the methods and systems are not limited tospecific methods, specific components, or to particular configurations.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Ranges may be expressed herein as from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, andindependently of the other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

Disclosed are components that can be used to perform the disclosedmethods and systems. These and other components are disclosed herein,and it is understood that when combinations, subsets, interactions,groups, etc. of these components are disclosed that while specificreference of each various individual and collective combinations andpermutation of these may not be explicitly disclosed, each isspecifically contemplated and described herein, for all methods andsystems. This applies to all aspects of this application including, butnot limited to, steps in disclosed methods. Thus, if there are a varietyof additional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

The present methods and systems may be understood more readily byreference to the following detailed description of preferred embodimentsand the Examples included therein and to the Figures and their previousand following description.

As will be appreciated by one skilled in the art, the methods andsystems may take the form of an entirely hardware embodiment, anentirely software embodiment, or an embodiment combining software andhardware aspects. Furthermore, the methods and systems may take the formof a computer program product on a computer-readable storage mediumhaving computer-readable program instructions (e.g., computer software)embodied in the storage medium. More particularly, the present methodsand systems may take the form of web-implemented computer software. Anysuitable computer-readable storage medium may be utilized including harddisks, CD-ROMs, optical storage devices, or magnetic storage devices.

Embodiments of the methods and systems are described below withreference to block diagrams and flowchart illustrations of methods,systems, apparatuses and computer program products. It will beunderstood that each block of the block diagrams and flowchartillustrations, and combinations of blocks in the block diagrams andflowchart illustrations, respectively, can be implemented by computerprogram instructions. These computer program instructions may be loadedonto a general purpose computer, special purpose computer, or otherprogrammable data processing apparatus to produce a machine, such thatthe instructions which execute on the computer or other programmabledata processing apparatus create a means for implementing the functionsspecified in the flowchart block or blocks.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including computer-readableinstructions for implementing the function specified in the flowchartblock or blocks. The computer program instructions may also be loadedonto a computer or other programmable data processing apparatus to causea series of operational steps to be performed on the computer or otherprogrammable apparatus to produce a computer-implemented process suchthat the instructions that execute on the computer or other programmableapparatus provide steps for implementing the functions specified in theflowchart block or blocks.

Accordingly, blocks of the block diagrams and flowchart illustrationssupport combinations of means for performing the specified functions,combinations of steps for performing the specified functions and programinstruction means for performing the specified functions. It will alsobe understood that each block of the block diagrams and flowchartillustrations, and combinations of blocks in the block diagrams andflowchart illustrations, can be implemented by special purposehardware-based computer systems that perform the specified functions orsteps, or combinations of special purpose hardware and computerinstructions.

In certain aspects, the accurate estimation of AVR can comprise one ormore of the following, optic disc detection, vessel segmentation, vesselwidth measurement, vessel network analysis, and artery/veinclassification. Optic disc detection can determine the location of theRegion of Interest (ROI) where the measurements are obtained. Vesselsegmentation can be used to find the vessels themselves and, dependingon the method that is used, the width of the vessels. The methods andsystems can then identify which vessels are arteries and which are veinswith high accuracy. Small classification errors can have a largeinfluence on the final AVR.

A protocol has been established for the measurement of the AVR. Theprotocol defines where and how measurements should be obtained. Theautomated methods and systems described herein follow this protocolwhenever possible. In an aspect, the methods and systems can bypre-process an image to remove the gradient around the border of thefield of view (FOV) as well as to remove slow intensity variations inthe image. Further pre-processing can be focused on the detection ofanatomical landmarks. These can comprise the optic disc, the landmark onthe retina around which the measurements are obtained and thevasculature, the structure that is actually being measured. Vessel widthmeasurements can be obtained and the vessels within the measurement areacan be classified into arteries and veins. Finally, the artery veinratio can be determined.

A. Preprocessing

The methods and systems can implement one or more preprocessingtechniques. For example, Field of View mirroring and background removal.Digital color fundus photographs have a black border around the FOV. Thelarge gradient can disturb feature measurements near the FOV border. Itcan be removed by applying a mirroring technique. This method mirrorspixel values from within the circular field of view to outside the FOV.This operation can be performed at the original image resolution. Slowbackground variations can be removed by blurring the image with aGaussian filter with a large standard deviation and subtracting theblurred image from the original. The value of the standard deviation ofthe Gaussian filter is not a critical parameter as long as it is largeenough to ensure the blurred image contains no visible structures suchas vessels. This procedure can be performed on both the Red as well asthe Green color planes separately. From here, whenever the Green and Redplane is mentioned, it refers to the pre-processed versions. The Bluecolor plane is not used.

In an aspect, pixel classification can be used to segment the retinalvasculature. The filter outputs of a Gaussian filter bank can be used asfeatures to train a kNN-classifier to detect the vasculature. Thismethod is not scale independent as the Gaussian filterbank features areextracted at particular scales. Additionally, the images with which thevessel segmentation method can be trained (for example, the DRIVEdatabase as is known to one of skill in the art) have a particularresolution and therefore a particular range of vessel widths (measuredin pixels). In a further aspect, images can be downsampled beforeapplying the vessel segmentation, for example with a factor 4.

In an aspect, the vessel segmentation method can assign each pixel inthe image a likelihood between 0 and 1 that the pixel is within avessel. This results in a “vesselness map” that can be thresholded toproduce a binary vessel segmentation. In an aspect, prior tothresholding the vesselness map can be up sampled back to the resolutionof the original image, for example by using quintic splineinterpolation. To analyze the vessel network a skeletonization methodcan be applied to the thresholded likelihood map, reducing all vesselsto a single centerline. In an aspect, the centerline can be one or morepixels wide. After the skeletonization of the segmented vessels,cross-over and bifurcation points can be removed by counting the numberof neighbors for all centerline pixels and removing those with more thantwo neighbors. This operation subdivides the vascular network into acollection of vessel segments that can be individually analyzed.

In an aspect, a supervised position regression method can be used todetect the centerpoint of the optic disc. This method can estimate howfar a certain position in the image is from the optic disc center. Thisestimation can be based on measurements obtained in the image and fromthe vessel segmentation. The target location can be found by obtainingestimates in many locations in the image, eliminating those locationsthat are estimated to be far from the optic disc and searching aroundthe locations estimated to be close to the optic disc center. The methodcan first be trained using a large set of images for which the locationof the optic disc is known. As this method does not provide an estimateof the size of the optic disc, a more or less constant size can beassumed. For example, a value of 360 pixels can be used for the diameterof the optic disc (DD). Other diameters can be used and determined bythose of skill in the art.

The AVR calculation protocol defines the region of interest (ROI) inwhich the AVR should be measured. This ROI is centered on the optic disc(see FIG. 1 ). FIG. 1 shows an image overlaid with the automaticallydetermined ROI region. The region labeled “B” is where AVR measurementsare taken. The ROI comprises several circular regions whose size isbased on the approximate diameter of the optic disc. Region A is between0.5 and 1 DD from the optic disc center and region B, where vesselmeasurements are taken, is between 1 DD and 1.5 DD from the optic disccenter. In an aspect, all analysis and evaluation performed by themethods and systems is within region B, however, it may be advantageousto expand or contract the region within which the methods and systemscan operate.

B. Vessel Width Measurement

In an aspect, after preprocessing, the vasculature can be thinned andsubdivided into a set of vessel segments. All vessel segments that arenot (partly) inside region B (FIG. 1 ) can be removed as they are notused in the AVR analysis. Even though the vessel segmentation methodprovided can successfully localize most vessels, i.e. wide and narrowones, choosing a single threshold to produce a binary segmentation ofthe vasculature that can be used to determine the local vessel width isdifficult. The values in the likelihood map, as produced by the vesselsegmentation method, tend to zero as one moves away from the vesselborder into the retinal background. This effect is also dependent on thevessel width with narrower vessels having an overall lower likelihoodresponse than wider vessels. Consequently, relatively small variationsin the applied threshold result in substantial vessel width differencesand thresholds that give visually pleasing results for wider vesselscompletely miss smaller vessels. This is further complicated by the factthat the vessel detection is performed at a lower resolution resultingin larger errors after upsampling.

In an aspect, a technique referred to as tobogganing can be combinedwith vessel segmentation. This has the added benefit that vessel widthanalysis can be performed on the original images in their originalresolution. Tobogganing is a segmentation technique that subdivides theimage into areas (i.e. “splats”) that are homogeneous based on a certaincriterion. The technique's results are analogous to the “catchmentbasins” in watershed segmentation. The multiscale gradient magnitudeimage can be used to define homogeneity. To calculate the gradientmagnitude, the image can be convolved with a first derivative ofGaussian filter in both the x and y direction after which the magnitudeof the gradient is obtained for each pixel. The gradient magnitude atvarious scales (i.e. various standard deviations σ) tends to have amaximum at the border of high contrast structures such as thevasculature. A lower scale filter can give more response at the borderof small vessels and a higher scale filter can give more response at theborder of wider vessels. To obtain the multiscale gradient magnitudeimage the scale-normalized gradient magnitude can be calculated atscales σ=1, 2, 3, 4, 5, 6 and the maximum value over scale for each ofthe pixels in the image can be used. After applying the tobogganingmethod, a splat map (see FIG. 2 c ) can be obtained. The likelihood mapproduced by the vessel segmentation algorithm can now be used todetermine for each splat the likelihood it is inside a vessel. This canbe accomplished by assigning to each splat the median likelihood valueof all the pixels that are part of the splat. It can be assumed thesplats are either inside or outside the vessel (note that thisassumption does not always hold in the case of low contrast, narrowvessels). Given a correct likelihood map, this results in the splatsinside the vessel being assigned a higher likelihood than those outsidethe vessel. In the resulting vessel map, the borders of the vessels arebetter defined and the widths of the vessels become less dependent onthe chosen vessel segmentation threshold. This enhanced vessel map canbe used to determine the vessel width. FIG. 2 shows the process in moredetail on a small subimage.

FIG. 2 is an image showing various processing steps described herein ona small sub-image. FIG. 2 a , the color fundus image. FIG. 2 b , thevessel likelihood map. A higher pixel value in this image means a highervessel likelihood. FIG. 2 c , the splat map, the borders of theindividual splats are shown. FIG. 2 d , the processed vessel likelihoodmap each splat has been assigned the median vessel likelihood valueunder the splat. FIG. 2 e , the vessel centerlines where each centerlinepixel has been assigned the likelihood it is inside a vein (dilated fordisplay). FIG. 2 f , the final width measures overlaid on the originalimage. Only every third measurement was plotted.

In an aspect, measurement of the local vessel width can be performedperpendicular to the local vessel angle in order to minimize errorsduring the measurement process. The local vessel angle can be determinedfor all centerline pixels in every vessel segment. The local vesselangle can be defined as the direction of the largest eigenvector of thecovariance matrix of the coordinate of the centerline pixel along withthe coordinates of its seven connected neighbors to both sides (i.e. 15coordinates in total). As it is unknown where the vessel begins or ends,the range of the angles is [0 . . . π]. Near the end of the vesselsegment only centerline coordinates inside the vessel are used, 8 forthe end pixel. Other values can be determined and used by those of skillin the art. FIG. 3 illustrates an artery vein centerline classificationresult (FIG. 3 b ) overlaid on a retinal image (FIG. 3 a ). A centerlinepixel with higher pixel intensity can have a higher likelihood to be avein.

In an aspect, for each centerline pixel, the local vessel width can bemeasured by finding the left and right vessel edges in the enhancedvessel map and calculating the distance between them. To determine thelocations of the edges of the vessel, the likelihood can be measuredalong a line through the centerline pixel and perpendicular to the localvessel angle. Starting from the centerline pixel, the vessel border canbe found in both left and right direction. The likelihood threshold atwhich a splat is no longer part of a vessel can be a critical parameterin this algorithm. As the likelihood assigned to vessel splats variesover the image and is dependent on local vessel contrast and vesselwidth, a local vessel threshold can be determined for every centerlinepixel. The vessel likelihood under the centerline pixel can bemultiplied with a ratio, for example 0.7, to determine the appropriatevalue for the vessel threshold.

In an aspect, after vessel widths for a vessel segment are determined,error correction can be performed by finding sudden, local changes inthe vessel width. A sudden change can be defined as more than apre-determined number of pixels from one centerline pixel to the other,for example, 3. A sudden change can happen when a vessel splat is not ora background splat is included in the vessel width measurement. Theratio threshold can be varied locally until the width measurement issimilar (<3 pixels difference) to the average width at the precedingvessel centerline pixels, for example 8 preceding vessel centerlinepixels. If the vessel width can not be adjusted to match the mean width,the originally detected vessel width can be used. FIG. 2 shows widthmeasurement results in a small sub-image. In addition to measuring thevessel width, the location of the left and right vessel boundary foreach centerline pixel can be stored. Using these two points, a profileacross the vessel can be defined and then used to extract feature datafrom across the vessel. FIG. 4 is a detail of a fundus photographshowing the measurement diameters and the vessel crossings where widthmeasurements are obtained. The white dots indicate the points on thevessels where measurements are obtained.

In a further aspect, a graph based approach can be used for vesselboundary delineation. A vessel centerline image can be derived from thevesselness map, and the two borders of the vessel can then be segmentedsimultaneously by transforming the first derivative of the vessel'spixels intensities into a two-slice three-dimension surface segmentationproblem. In retinal images, the boundaries of the blood column form areliable proxy for vessel diameter. While the splat based approachprovides reliable results, a graph-based approach to determine thelocation of the vessel boundary can provide greater speed and accuracy,as graph-based approaches are known to be globally optimal. In additionto AVR analysis, automated determination of vessel width measurementbased on segmentation of both vessel boundaries can also allow thegeometry of the retinal vessels to be quantified, as the geometry isalso affected by cardiovascular disease, diabetes, and retinal disease.Finally, accurate determination of the vessel boundary may allow localpathologic vascular changes such as tortuosity and vessel beading to bemeasured accurately.

In order to detect both boundaries simultaneously, a two-slice 3-D graphcan be built and a smoothness constraint between the two slices can beapplied. Thus, a simultaneous two-D boundary segmentation is transformedinto a two-slice 3-D surface segmentation problem. This problem is thenfurther converted into the problem of computing a minimum closed set ina node-weighted graph.

An initial segmentation is needed to build the graph. The vesselness mapcan be used as the initial segmentation. One example image is shown inFIG. 5 . FIG. 5 a is a color fundus image. The white rectangular isenlarged and used for illustration in FIG. 6 . FIG. 5 b is thecorresponding vesselness image. By thresholding the gray scale image, abinary vessel segmentation can be generated. A (constant) low thresholdcan be selected to better maintain the continuity of blood vessels. Thetrade-off is that small regions of noise may not be suppressedadequately. In order to solve this problem, the vessel regions with anarea smaller than a pre-determined number (for example, 20) pixels canbe erased from the thresholded image. A sequential thinning approach canthen be applied to the binary vessel segmentation to find the vesselcenterlines. In an alternative aspect, vessel width can be measureddirectly from the vesselness map. The vesselness image can bethresholded, generating a binary vessel segmentation. By way of example,a fixed threshold of 190 can be used. Widths can then be measured onthis binary vessel map.

From the vessel centerline image, the bifurcation points and crossingpoints can be excluded. A bifurcation point can be defined as acenterline pixel with three eight-connected neighbors and a crossingpoint can be defined as a centerline pixel with four or moreeight-connected neighbors. Hence, the vessel centerline image can bescanned first to get the centerline pixels that have three or moreneighbors. By deleting the bifurcation points and crossing points, thevessel trees can be cut into vessel segments. From these vesselsegments, the end points which have only one neighbor pixel can be foundby scanning the image again. Starting from one end point, the vesselsegment can be traced until the other end point is reached. In this way,all the vessel segments on the image are traced and labeled.

For each labeled vessel segment, the growing direction for everycenterline pixel can be calculated. n adjacent centerline pixels can beused on both sides of the target centerline pixel and principalcomponent analysis can be applied on the resulting vessel segment. Thevalue of n can be a parameter of the image size, which is approximately0.005 times the first dimension of the image size. For example, if thefirst dimension of an image is 600 pixels, the value of n would be threepixels, resulting in a total vessel segment length of seven pixels. Inan aspect, the minimum value of n can be two. The first principalcomponent can correspond to the growing direction of the pixel. An endpoint that does not have enough neighboring centerline pixels to definethe vessel growing direction can be determined to have the direction ofthe nearest centerline pixel that has a definition of a vessel growingdirection.

For the vessel growing direction, a counter-clockwise 90 degree isregarded as the normal direction for this point. Using the centerlinepixels as the base nodes, profiles on the positive direction of thenormals can be built as one slice and profiles on the negative directioncan be built as another slice, as shown in FIG. 6 .

FIG. 6 is an illustration of how to luxate the vessel normal profilesinto a graph. FIG. 6 a , a small segment of vessel extracted from FIG. 5. FIG. 6 b , the vessel probability image of FIG. 6 a . FIG. 6 c , thevessel centerline image of FIG. 6 a . FIG. 6 d , the vessel growingdirections are calculated and node normal profile perpendicular to thevessel growing direction are constructed. FIG. 6 e , red normal profilesin FIG. 6 d are used to build the red graph slice and the green normalprofiles in FIG. 6 d are used to build the green graph slice. The blacknodes represent base nodes, corresponding to the black centerline pixelsin FIG. 6 d . Consequently, the red slice represents one boundary inFIG. 6 d and the green slice represents the other boundary in FIG. 6 d .Smoothness constraints (controlled by the arcs between adjacent columns)are applied differently to control the boundary smoothness within oneboundary and between the two boundaries.

Along each normal profile Col (x, y) every node V (x, y, z)(z>0) has adirected arc to the node V(x, y, z−1). Along the x-direction, meaningalong the same boundary, for each node, a directed arc is constructedfrom V (x, y, z)∈Col (x, y) to V (x+1, y, max(0, z−Δ_(x)))∈Col (x+1, y).Similarly, arcs from V (x, y, z)∈Col (x, y) to V (x−1, y, max(0,z−Δ_(x)))∈Col (x−1, y, z) are constructed. Δ_(x) is the maximumdifference allowed between two adjacent normal profiles within oneboundary. Along the y-direction, meaning between the two slices, arcsfrom V (x, y, z)∈Col (x, y) to V (x, y+1, max (0, z−Δ_(y)))∈Col (x, y+1,z) and arcs from V (x, y, z)∈Col (x, y) to V (x, y−1, max (0,z−Δ_(y)))∈Col (x, y−1, z) are constructed. Δ_(y) is the maximumdifference allowed between two corresponding normal profiles between thetwo boundaries. The base nodes are all connected. A surface is regardedfeasible if it satisfies the smoothness constraint defined by Δ_(x) andΔ_(y). An optimal surface is defined as the surface with the minimumcost among all feasible surfaces defined in the 3-D volume.

The cost image can be generated from the orientation sensitive 1D firstorder derivative of Gaussian of the green channel. The green channel ofthe color image usually shows the highest contrast between the bloodvessels and background. In order to find the gradient of vesselsspecified at different locations with different orientations, a discreteconvolution with the 1D derivative of Gaussian can be applied along thedirection perpendicular to the vessel growing direction (as shown inFIG. 7 ). The derivative of Gaussian kernel is shown in Equation 1:

$\begin{matrix}{{{f\left( {x;\sigma} \right)} = {{- \frac{x}{\sigma^{3}\sqrt{2\pi}}}{\exp\left( {- \frac{x^{2}}{2\sigma^{2}}} \right)}}},} & (1)\end{matrix}$where σ is the only parameter in the first order derivative of Gaussian.FIG. 7 is an example. FIG. 7 a shows an enlarged part of the greenchannel image. FIG. 7 b shows the orientation sensitive 1D derivative ofGaussian result.

The value of Δ_(χ) can be set to one to maintain the smoothness withinone boundary and Δ_(y) can be set to two so that a difference is allowedbetween widths from the centerline to the left edge and from thecenterline to the right edge, in case the centerline is not exactly atthe center of the two edges. After the node-weighted directed graph isconstructed, the optimal surface can be determined. To determine thevessel width, the coordinate difference between the two correspondingnodes on the optimal surface from the two slices can be calculated. Toshow the boundary on the image, the nearest integer coordinate of everynode on the optimal surface can be calculated and shown on the image.

C. Classification into Arteries and Veins

To enable separate analysis of the arteries and the veins in the image,the previously detected vessel segments can be assigned to one of thesetwo classes. In an aspect, a supervised system, i.e. trained withexamples, can be used. After a training procedure, the classificationmethod can be used to classify previously unseen centerline pixels intoeither artery or vein (AV classification). The pre-processing procedureand vessel width measurements can be applied to all images in thetraining set. An expert can indicate whether a given major vessel was anartery or vein for each of the training images.

In the training phase, a classifier can be trained using the expertlabeled vessels in the training set in order to distinguish between bothclasses of centerline pixels. As not all vessels in the training setwill be marked as artery or vein, centerline pixels from unmarkedvessels are not included in the training dataset. For all remainingcenterline pixels in the training images, a set of local features wasextracted. Table I shows a list of exemplary extracted features that canbe used. In general the features characterize the color as well as thecolor variation in the vessel. All features that are measured across thevessel can be measured on the profiles as previously determined.

TABLE 1 Nr. Feature description 1-3 Normalized Mean Hue, Saturation andIntensity across vessel. 4-5 Normalized Mean Red and Green planeintensities across the vessel. 6-8 Standard deviation of Hue, Saturationand Intensity across the vessel.  9-10 Standard deviation of Red andGreen plane intensities across the vessel. 11-13 Normalized Hue,Saturation and Intensity under the centerline pixel. 14-15 NormalizedRed and Green plane intensity under the centerline pixel. 16-19Normalized highest and lowest intensity in the Red and Green planeacross the vessel. 20-27 Intensity under the centerline pixel in aGaussian blurred (σ = 2, 4, 8, 16) version of the Red and Green plane.

The absolute color of the blood in the vessels can vary between imagesand across subjects. This variation can have several causes. Primarily,the amount of hemoglobin oxygen saturation influences the reflectivityof the blood column, and this difference allows the difference betweenhigher saturation arterial from lower saturation venous blood to bevisualized. Next, lens absorption for different wavelengths isinfluenced by aging and the development of cataract, causing shifts inthe spectral distribution of light reflected by blood. Individualdifference in pigmentation of the retinal pigment epithelium below theblood vessels also influence the spectrum of reflected light. Finally,across examinations, even from the same subject, differences in flashintensity, flash spectrum, nonlinear optical distortions of the camera,flash artifacts, and focus also cause considerable variability. Thesefactors complicate classification substantially, and normalization tozero mean and unit standard deviation of the vessel color features forevery individual image can improve classification. After sampling thefeatures for each centerline pixel, the appropriate labels can beassigned based on the reference standard and all training samples can bestored in a training dataset. This sampling process can be repeated forall images in the training set.

The training set can then be split into a separate classifier selectiontraining and test set. Several different classifiers can be used, forexample: k-Nearest Neighbor Classifier, Support Vector MachineClassifier, Linear Discriminant Classifier, or a Quadratic DiscriminantClassifier. The classifier that maximizes the area under the ReceiverOperator Characteristic (ROC) curve can be selected.

After the training phase is complete, the trained classifier can beapplied to the images in the test set. All the test images can bepreprocessed similarly to the training images. For each centerline pixelthe complete set of 27 features was extracted, however fewer and/ordifferent features can be used. The trained classifier can then be usedto assign a soft label l=[0 . . . 1]. Here, a label close to 0 means thepixel was likely in an artery and a label close to 1 means a pixel waslikely in a vein. It can be assumed that all pixels in a vessel segmentare either in an artery or a vein. Each soft label assigned to acenterline pixel can be regarded as a vote for the label of the completesegment. Combining these votes can be done in many different waysincluding taking the median label for the entire vessel segment.

Due to variation in the local image characteristics, the soft labelsassigned to each of the segments can vary over the image. A globalthreshold will not always successfully separate the arteries from theveins within a single subject and will vary between subjects. To performthe final classification the prior knowledge that arteries and veinsusually come in pairs can be used. This means that, when going aroundthe optic disc in a circle in region B of the ROI, one will generallyencounter an artery after first encountering a vein and vice versa.Since this rule does not always hold and since finding the matchingvessel segment for a particular different vessel segment is non-trivial,a voting procedure can be implemented.

During a voting procedure, all vessel segments intersecting with acircle of a certain diameter around the optic disc and within region Bof the ROI can be eligible for matching. Finding the nearest neighborvessel segment on a circle is straightforward and can be done by findingthe nearest intersection point on the circle. The soft AV labels of bothpoints can be compared and the vessel segment with the highest softlabel can receive a vote for “vein” and the other can receive an“artery” vote. Then the next nearest unpaired vessel can be selected andthe procedure repeated. The outcome of this procedure is dependent onthe starting vessel segment. By picking a different vessel segment asthe starting segment, the distribution of the AV votes amongst thevessel segments will vary. All vessel segments eligible for matching cantherefore be selected once as the starting vessel and the matchingprocedure repeated. Finally, the votes can be counted and each of thevessel segments assigned a hard label (i.e. either artery or vein basedon the received votes). Vessel segments with an equal number of arteryand vein votes can be excluded from analysis.

D. Determining the AVR

In an aspect, the arteriolar-to-venular ratio can be defined as

${AVR} = \frac{CRAE}{CRVE}$where CRAE is the Central Retinal Artery Equivalent and CRVE is theCentral Retinal Vein Equivalent. To calculate these numbers, aniterative process for matching up vessels and calculating the CRAE andCRVE can be used. The widest 6 veins and arteries (these do not have tobe paired) can be used although fewer total number of widths can be usedin case not enough measurement points are available. Algorithm 1 showsan exemplary implementation.

Algorithm 1 The algorithm used to calculate the AVR Input: Vector A oflength |A| containing the widths of the found arteries and vector V oflength |V| containing the widths of the found veins. Output: The AVR.Sort A and V in decreasing order and set the length of both vectors tomin(|A|, |V|, 6). while |A| > 1 do Select and remove the first element fand last element l from A Store √{square root over ((f² + l²) * 0.88)}in vector C if |A| equals 1 then Remove the last remaining element fromA and store it in C end if A = C Sort A from large to small end whileClear C while |V| > 1 do Select and remove the first element f and lastelement l from V Store √{square root over ((f² + l²) * 0.95)} in vectorC if |V| equals 1 then Remove the last remaining element from V andstore it in C end if V = C Sort V from large to small end while Dividethe remaining clement in A by the remaining element in V to obtain thefinal AVR.

In an aspect, the final voting procedure can be based on measurementsobtained on a circle with a certain diameter. The chance that all vesselsegments in the ROI will intersect with a circle of any particulardiameter is small. Therefore, the voting procedure and AVR calculationshould be repeated at various diameters within the AVR ROI (see FIG. 4). By way of example, the diameters can be from 1 DD to 1.5 DD in stepsof 0.1 DD where DD was 360 pixels, so the voting and AVR calculationprocedure can be repeated 6 times. Note that this samples the AVR ROIequidistantly. For each circle diameter, the AV voting procedure can beperformed, the local vessel width can be measured and stored in twovectors, A for arteries and V for veins. Next, Algorithm 1 can be usedto calculate the AVR. The resulting six AVR values can be averaged toarrive at the final AVR estimate for the complete image.

In an aspect, illustrated in FIG. 8 , provided are methods and systemsfor automatic determination of arteriovenous ratio (AVR), comprisingreceiving an image at 801, detecting a region of interest in the imageat 802, identifying arteries and veins in the region of interest at 803,determining vessel width measurements for the identified arteries andveins at 804, and estimating an arteriovenous ratio (AVR), from thevessel width measurements at 805. In an aspect, decreased AVR indicateshigher propensity for a disease. For example, decreased from a “normal”AVR or decreased from a previous AVR. Thus, the methods and systemsprovide a novel quantitative approach to early detection of retinal andcardiovascular disease.

In an aspect, detecting a region of interest in the image can furthercomprise detecting an optic disc in the image. Identifying arteries andveins in the region of interest can comprise performing vesselsegmentation on the image. Performing vessel segmentation on the imagecan further comprise utilizing a trained classifier to classify pixelsin the image as artery or vein pixels.

In an aspect, the image can be one or more of, a color image, amultispectral image, an Optical Coherence Tomography image. The imagecan be of the retina, iris, skin, brain surface, or any tissue withvisible blood vessels imaged using any two different wavelength imagingprocess.

The trained classifier can use a feature vector comprising one or moreof, derivatives, texture, and color properties.

In an aspect, determining vessel width measurements for the identifiedarteries and veins comprises using one or more of, a graph search, amultiscale pixel feature based tobogganing method and splats. The graphsearch can use a multiscale cost function. The multiscale cost functioncan be derived from some combination of wavelet kernel lifting includingGabor, Gaussian derivative and Difference of Gaussians kernels.

The methods and systems provided can be applied to other two-dimensionalblood vessel projections such as cardiac and brain angiograms.

In a further aspect, the methods and systems can be utilized to providean indicator of disease propensity. The methods and systems can beapplied to large quantities of images for batch processing anddetermination of a disease propensity for a group of persons.

In an aspect, provided are methods and systems to automaticallydetermine artery/vein measures in an image region in an image. Themethods and systems can utilize disc detection, disc centerpointdetermination and disc diameter measurement to identify the imageregion. Pixel classification and mathematical morphology can be used todetermine the artery/vein measures. The image can be a color image. Theimage can be a multispectral image. The image can be a combination of anOCT image and a multispectral image. The image can be of the retina, theiris, the skin, the brain surface, or of another tissue with visibleblood vessels imaged using any two different wavelength imaging process.

In an aspect, the methods and systems can classify pixels in an imageusing artery and vein measurements. The classification can use a featurevector containing derivatives, texture and color properties. The colorfeatures can be normalized for each individual image. By way of example,a knn classifier or a support vector machine can be used to classify thepixels. The centerline of one or a plurality of vessels can be assigneda classification.

In a further aspect, provided are methods and systems to accuratelymeasure local vessel parameters to supply features to the methodsdisclosed. In a further aspect, the methods and systems can measurelocal vessel texture patterns using filters. The filters can be, forexample, Gaussian derivatives, Gabor filters or wavelet filters basedfilterbank, or a combination thereof. The filters can be obtained usingimage samples and a transform such as principal component analysisthereof.

In a still further aspect, provided are methods and systems to propagatepixel based artery vein classifications over the complete vessel toclassify the entire vessels themselves. In a further aspect, providedare methods and systems to perform accurate width measurements of theretinal vessel between the boundaries of the vessel. The vesselboundaries can be determined using graph search. The graph search canuse a multiscale cost function. The multiscale cost function can bederived from some combination of wavelet kernel lifting including Gabor,Gaussian derivative and Difference of Gaussians kernels. The boundariescan be determined using a multiscale pixel feature based tobogganingmethod and splats.

In an aspect, the methods and systems disclosed can analyze local vesselstructure to determine how the vascular network is connected. In afurther aspect, the methods and systems disclosed can determine thelocations in the image where width measurements need to be obtained inorder to calculate the AV-ratio. Disc detection, disc centerpointdetermination and disc diameter measurement can be used to identify theimage locations. The features can be normalized to all vessel pixels.The features can be normalized to all disc vessel pixels. The featurescan be normalized to all background pixels.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the scope of the methods and systems. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.), but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. or is at ambient temperature, and pressure is at or nearatmospheric.

In a first experiment, sixty-five digital color fundus photographs wereacquired for training and testing. All images were obtained frompatients with primary open angle glaucoma at the University of IowaHospitals and Clinics using a 30° degree Zeiss fundus camera (Carl ZeissMeditec, Dublin, Calif.), with digital back (OIS systems, Sacramento,Calif.). The images were centered on the disc. The dimensions of theimages are 2392×2048 pixels with 8-bits per pixel per color plane, andstored in JPEG format for export. To train the AV classificationcomponent and determine the parameters for the algorithm, 25 digitalcolor fundus photographs were randomly selected from the set of 65. Theremaining 40 images were assigned to the test set and were only used toevaluate the complete system.

An ophthalmologist (AVD) labeled the major vessels in the images of thetraining set as either artery or vein to train the artery veinclassification method. As only the vessel centerlines needed to belabeled, precise vessel segmentation was not needed. Labeling was doneby manually drawing a line over the major vessels using a standardpainting program. The colors blue and red were used for veins andarteries respectively.

Two components of the presented method were evaluated on the testimages, the AV classification and the AVR determination. To evaluate theAV classification, the vessels in each of the images in the test setwere manually labeled as either artery or vein by an ophthalmologist(AVD). In contrast to the way the major arteries and veins were labeledin the training set, only those parts of all vessels (i.e. including thesmall vessels) that were inside the AVR ROI were labeled in the testset. The expert labeled all vessels in the ROI as either an artery or avein.

To set the AVR reference standard, a semi-automated computer programdeveloped by the University of Wisconsin in Madison, Wis., USA was used(IVAN). Two ophthalmologists processed the images in the test set usingthis software. Both ophthalmologists were instructed in the use of thesoftware using the protocols defined by the software developers. IVAN issemi-automated; the AVR ROI localization, the vessel width measurementsand the artery vein classification require manual adjustment. On averagea human observer takes around 10 minutes per image to perform theanalysis. The software is capable of producing several different AVRmeasures. The ratios obtained by the first ophthalmologist (MDA) wereused as the reference standard and the second ophthalmologist's (AVD)ratios were used to determine the variability between experts.

The methods and systems provided herein were applied to all 40 testimages. The system was able to find the optic disc and successfullyplaced the AVR in all 40 images. This was verified by visual inspectionand the AVR ROI was centered on the optic disc in all 40 images.

To compare the artery/vein classification with the labeling by the humanexpert we performed an ROC analysis with class 0 being artery and class1 being vein. Note that this analysis was performed on the vesselcenterline pixels only. The proposed classification system assigned eachcenterline pixel a likelihood value that it was inside of a vein. Thatis, a value of 0 indicates a centerline pixel likely inside an arteryand a value of 1 indicates a centerline pixel likely in a vein. A ROCanalysis was performed and the resulting curve is shown in FIG. 9 . Thesystem attained an area under the curve of 0.84. This means that, givena randomly selected artery pixel and a randomly selected vein pixel, in84% of cases the automatic system will correctly assign a higherlikelihood to the vein centerline pixel.

To evaluate the ability of the system to assign an artery vein ratio toan image the AVRs as produced by the system and the second observer weredirectly compared with the reference standard. Paired t-test showed thatthere was no significant difference between the reference standard andthe system's measurements (p=0.66). The same holds for the secondobserver's measurements (p=0.59). Table 2 shows the results for theindividual image. The “Reference” column contains the reference standardfor reading, “System” is the output from the automatic system, and “Obs.2” contains the reading from the second observer. All number in italicsrepresent differences between the first and second. To visually assessthe agreement between both the automatic system and the second observerand the reference standard the results are plotted in Bland-Altman plotsin FIG. 10 . This graph plots the mean of two AVR measurements againstthe difference between them and allows a visual assessment of thedistribution of errors and the agreement between the two methods. FIG.10 provides the Bland-Altman plots or the agreement between theautomatic system and the reference standard FIG. 10 a and between thesecond observer and the reference standard FIG. 10 b . The red linesrepresent the 95% limits or agreement. The dotted line represents themean difference between AVR measurements.

TABLE 2 Image Reference System Error Obs. 2 Error  1 0.70 0.62 0.08 0.710.01  2 0.76 0.81 0.05 0.75 0.01  3 0.66 0.81 0.15 0.69 0.03  4 0.750.76 0.01 0.75 0.00  5 0.53 0.59 0.06 0.61 0.08  6 0.93 0.80 0.13 0.760.17  7 0.63 0.70 0.07 0.68 0.05  8 0.70 0.59 0.11 0.65 0.05  9 0.650.68 0.03 0.64 0.01 10 0.78 0.74 0.04 0.75 0.03 11 0.65 0.58 0.07 0.650.00 12 0.67 0.74 0.07 0.65 0.02 13 0.64 0.68 0.04 0.71 0.07 14 0.690.77 0.08 0.76 0.07 15 0.56 0.57 0.01 0.85 0.23 16 0.64 0.70 0.06 0.740.10 17 0.57 0.60 0.03 0.62 0.05 18 0.62 0.59 0.03 0.58 0.04 19 0.640.67 0.03 0.61 0.03 20 0.68 0.67 0.01 0.68 0.00 21 0.52 0.55 0.03 0.450.07 22 0.62 0.58 0.04 0.63 0.01 23 0.67 0.71 0.04 0.63 0.04 24 0.710.67 0.04 0.62 0.09 25 0.57 0.59 0.02 0.58 0.01 26 0.72 0.74 0.02 0.760.04 27 0.65 0.63 0.02 0.64 0.01 28 0.56 0.69 0.13 0.49 0.07 29 0.73 0.60.13 0.61 0.12 30 0.64 0.66 0.02 0.63 0.01 31 0.63 0.61 0.02 0.68 0.0532 0.72 0.68 0.04 0.70 0.02 33 0.59 0.61 0.02 0.61 0.02 34 0.61 0.710.10 0.59 0.02 35 0.65 0.68 0.03 0.61 0.04 36 0.74 0.59 0.15 0.64 0.1037 0.69 0.64 0.05 0.62 0.07 38 0.82 0.72 0.10 0.79 0.03 39 0.61 0.700.09 0.64 0.03 40 0.74 0.81 0.07 0.62 0.12 mean 0.67 0.67 0.06 0.66 0.05SD 0.08 0.07 0.04 0.08 0.05 min 0.52 0.55 0.01 0.45 0.00 max 0.93 0.810.15 0.85 0.29

This study showed that a completely automatic method can estimate theAVR in retinal color images with a mean error similar to that of a humanexpert who was using the reference standard system IVAN. The automaticmethod also successfully classified retinal vessel pixels into beingpart of an artery or vein.

Compared to previously presented methods the area under the ROC curvefor the AV classification may not seem an improvement. However, it isimportant to note that, in contrast with previously presented methods,all detected vessel centerline pixels inside region B of the ROI wereclassified. This includes vessels for which the observers were not ableto see whether they were an artery or a vein without tracing the vesselback to its source. The prior methods have used clustering instead ofclassification to overcome the challenges presented by the highvariability between fundus images. The use of color features that arenormalized for each individual image, combined with a supervisedclassification approach, leads to better results than using clustering.

Table 2 shows the error with respect to the reference standard of theautomated system for each image. The mean AVR values and their standarddeviations of the reference standard are very close between theautomated system and observer 2, a statistical test showed there was nosignificant difference between the means. Nevertheless, there are 8 AVRmeasurements by the automated system that have an error above 0.10 whencompared with the reference standard. However, of these, 5 show arelatively good agreement with the measurement done by the secondobserver. The Bland-Altman plots (see FIG. 10 ) show that both theautomated system and the second observer have no bias as the meandifference between the AVR measurements is close to 0. The 95% limits ofagreement for both the automated and second observer are also almost thesame. However, the second observer has two large outliers without whichthe 95% limits of agreement would have moved somewhat closer to 0.

In a second experiment, to evaluate our method further, the relationshipbetween the average vessel width (both arteries and veins) and thedistance to the optic disc was determined in a sample of 600 compositecolor fundus images, each consisting of two registered and blendedfundus images.

The REVIEW database was used to validate the correctness of the methodsand systems. Each profile in the REVIEW database consists of 15 numbers:series number, image number, segment number and four data for each ofthree observers. The three observers are denoted as O₁, O₂ and O₃. Foreach observer, there are four measurements for two points on each edge(x₁, y₁, x₂ and y₂). The vessel centerline is defined as(x₁−x₂)²+(y₁−y₁)² and the vessel width as (x₁−x₂)²+(y₁−y₂)² for eachobserver (http://ReviewDB.lincoln.ac.uk). A reference standard, denotedby RS, is then created by averaging the manual results of the threeobservers.

The vessel centerlines were determined as described herein and mapped tothe centerlines in the RS, so that only those vessel centerline pixelswere analyzed that also occurred in the RS. Otherwise the centerlinepixel was labeled as absent.

The methods and systems described herein were compared with the resultsof prior methods, where 1DG and 2DG mean 1-D Gaussian model-fitting and2-D Gaussian model-fitting, respectively.

To determine this relationship on the dataset of 600 registered fundusimages the vessel widths of all vessels was obtained as describedherein. Circular regions of interest one pixel wide were centered on theoptic disc center. The average width of all vessels in each region ofinterest (i.e. both arteries and veins) was then plotted in a graph, aswell as the 95% confidence interval (CI) of the average width.Centerline pixels with a distance larger than 450 pixels from the discwere too close to the edge of the image—less than 2.1% of all centerlinepixels—and were eliminated from further analysis.

An example of the quality of the obtained vessel boundary detection isshown in FIG. 11 . Good performance is obtained on even the smallestvessels, which have low contrast. FIG. 11 a is one image from the CLRISdatabase. Image size is 2160×1440, a=4. FIG. 11 b is an enlarged part ofimage FIG. 11 a.

The methods and systems provided herein were compared to other knownalgorithms in Table 3 to Table 6. The first three rows are the manualresult from the observers. Rows 4-8 show the results of the otheralgorithms. The last row shows the result of the presently disclosedmethods and systems. The first column is the success rate. Everycenterline pixel that had corresponding ground truth profiles as a“success measurement”, irrespective of the estimate of the width.Therefore all measurements with a corresponding ground truth profilewere included even if the measurements did not result in a meaningfulestimation. The columns 2-3 are the mean and standard deviation of allmeasurements labeled as ‘success measurement’. Columns 4-5 are thesigned mean and standard deviation of the point-by-point differences.

TABLE 3 VESSEL WIDTH MEASUREMENT ACCURACY OF HRIS (SUCCESS RATE INPERCENTAGE, MEAN μ AND STANDARD DEVIATION σ IN PIXEL) Method SuccessMeasurement Difference Name Rate % μ σ μ σ Observer 1 (O₁) 100 4.12 1.25−0.23 0.288 Observer 2 (O₂) 100 4.35 1.35 0.002 0.256 Observer 3 (O₃)100 4.58 1.26 0.23 0.285 Gregson's Algorithm 100 7.64 — 3.29 2.841Half-height full-width 88.3 4.97 — 0.62 0.926 (HHFW) 1-D Gaussian Model-99.6 3.81 — −0.54 4.137 fitting 2-D Gaussian Model- 98.9 4.18 — −0.176.019 fitting Extraction of Segment 99.7 4.63 — 0.28 0.42 Profiles (ESP)Proposed Method 100 4.56 1.30 0.21 0.567

TABLE 4 VESSEL WIDTH MEASUREMENT ACCURACY OF CLRIS (SUCCESS RATE INPERCENTAGE, MEAN μ AND STANDARD DEVIATION σ IN PIXEL) Method SuccessMeasurement Difference Name Rate % μ σ μ σ Observer 1 (O₁) 100 13.194.01 −0.61 0.566 Observer 2 (O₂) 100 13.69 4.22 −0.11 0.698 Observer 3(O₃) 100 14.52 4.26 0.72 0.566 Gregson's Algorithm 100 12.8 — −1.0 2.841Half-height full-width 0 — — — — (HHFW) 1-D Gaussian Model- 98.6 6.3 —−7.5 4.137 fitting 2-D Gaussian Model- 26.7 7.0 — −6.8 6.019 fittingExtraction of Segment 93.0 15.7 — −1.90 1.469 Profiles (ESP) ProposedMethod 94.1 14.05 4.47 0.08 1.78

TABLE 5 VESSEL WIDTH MEASUREMENT ACCURACY OF VDIS (SUCCESS RATE INPERCENTAGE, MEAN μ AND STANDARD DEVIATION σ IN PIXEL) Method SuccessMeasurement Difference Name Rate % μ σ μ σ Observer 1 (O₁) 100 8.50 2.54−0.35 0.543 Observer 2 (O₂) 100 8.91 2.69 0.06 0.621 Observer 3 (O₃) 1009.15 2.67 0.30 0.669 Gregson's Algorithm 100 10.07 — 1.22 1.494Half-height full-width 78.4 7.94 — −0.91 0.879 (HHFW) 1-D GaussianModel- 99.9 5.78 — −3.07 2.110 fitting 2-D Gaussian Model- 77.2 6.59 —−2.26 1.328 fitting Extraction of Segment 99.6 8.80 — −0.05 0.766Profiles (ESP) Proposed Method 96.0 8.35 3.00 −0.53 1.43

TABLE 6 VESSEL WIDTH MEASUREMENT ACCURACY OF KPIS (SUCCESS RATE INPERCENTAGE, MEAN μ AND STANDARD DEVIATION σ IN PIXEL) Method SuccessMeasurement Difference Name Rate % μ σ μ σ Observer 1 (O₁) 100 7.97 0.470.45 0.234 Observer 2 (O₂) 100 7.60 0.42 0.08 0.213 Observer 3 (O₃) 1007.00 0.52 0.52 0.233 Gregson's Algorithm 100 7.29 — −0.23 0.602Half-height full-width 96.3 6.47 — −1.05 0.389 (HHFW) 1-D GaussianModel- 100 4.95 — −2.57 0.399 fitting 2-D Gaussian Model- 100 5.87 —−1.65 0.337 fitting Extraction of Segment 100 6.56 — −0.96 0.328Profiles (ESP) Proposed Method 99.4 6.38 0.59 1.14 0.67

Among the four datasets, the HRIS dataset has the highest imageresolution (3584×2438 pixels). The performance of the vessel widthdetection is comparable to the observers.

The CLRIS dataset images have a resolution of 2160×1440 pixels. The meanvessel with and the point-by-point difference mean are very close to theobservers' performance. But the point-by-point difference standarddeviation is high. The VDIS dataset has a resolution of 1360×1024pixels. The mean vessel width is 0.53 pixels smaller than the groundtruth. The KPIS dataset has the smallest image resolution, 288×119pixels and 170×92 pixels.

Testing indicates that fundus images with a higher resolution can bemeasured more accurately with a larger σ. The final choice of a is σ=7for HRIS dataset, σ=4 for CLRIS and VDIS, σ=1 for KPIS.

FIG. 12 shows the correlation of predicted vessel width and the mean ofthe observers' measurement for HRIS. The regression line shows theprediction tends to give a smaller measurement for vessels with finevessels while a larger measurement for large vessels, compared to theobservers' measurements. Each point represent one profile. The red lineis the y=x line. The black line is the regression line (y=0.87x+1.56).

The relationship between vessel width and distance from the optic disccenter is shown in FIG. 13 . A total of 2,936,749 center line pixelswere extracted from the 600 images. If the pixels near the center ofoptic disc are not considered, where the blood vessels show a verycomplicated structure, the average vessel width shows a monotonicdecrease from a distance of 20 pixels to 450 pixels, with a sudden slopechange at a distance of 300 pixels to the optic disc. For vessels nearthe optic disc border, the average vessel width is about 5.0 pixels(approximately 75 microns) while the vessels that have a distance of 450pixels to the optic disc have an average vessel width of 3.5 pixels(approximately 52.5 microns).

FIG. 13 illustrates relationship between vessel width and distance tooptic disc. The black circle marks a 400 pixels to the optic disccenter. The vessel width analysis starts from the optic disc center andends at a distance of 450 pixels (the x-axis). The y-axis shows thevessel width in pixels. The green line is the average vessel width. Theblack lines are the 95% confidence interval lines. Image resolution isabout 800×700 pixels. Scale parameter (J=4 pixels was used.

The methods and systems provided can identify the retinal blood vesselboundaries using a graph-based approach. The results of the evaluationshow that the blood vessel boundaries are accurate, and that the methodsand systems outperform the prior algorithm on two datasets of standardretinal image resolution. The presently disclosed methods and systemsallow for the identification of retinal vessel boundaries of even smallvessels on standard posterior pole images as used in diabeticretinopathy screening programs around the world.

The results indicate that the relationship between vessel width withdistance and the optic disc center is inverse and monotonous. Using thismethod, the relationship between the average retinal vessel diameter andthe distance from the optic disc from 600 patients with diabetes wasreliably determined. Except for the vessel pixels near the center of theoptic disc (about 20 pixels), the blood vessel width shows a monotonicdecrease considering the distance from the optic disc to the center.This is most likely caused by branching—if it is assumed that the bloodvessel volume to be unchanged. If the number of blood vessels is keptconstant, the higher the branch frequency with increasing distance tothe optic disc the larger the slope of the width decrease. At a distanceof around 300 pixels, there is a slight slope change in average vesselwidth, most likely caused by the fact that this is where the imagecentered on the optic disc ends and transitions to the fovea centeredimage that is registered to it in our dataset.

FIG. 14 illustrates vessel width measurement on vessel segments withdifferent resolutions, FIG. 14 a , one test vessel segment from HRIS.The length of the vessel segment is 173 pixels. FIG. 14 b one testvessel segment from KPIS. The length of the vessel segment is 226pixels. FIG. 14 c the vessel width measurement result of (a). If thedetected edge is not at an integer location, the nearest integercoordinate is shown. FIG. 14 d the vessel width measurement result of(b). If the detected edge is not at an integer location, the nearestinteger coordinate is shown. FIG. 14 e the cross-sectional view ofvessel intensity with regard to the distance to the centerline for (a).The black curve is the average of 173 normal profiles in the vesselsegment. Intensities at non-integer locations are linearly interpolated.The red star is the average vessel width across the whole vessel segmentmarked by observers. The green star is the average vessel width acrossthe whole vessel segment measured by proposed method. The two boundariesare flipped and shown in one figure. FIG. 14 f the cross-sectional viewof vessel intensity with regard to the distance to the centerline forFIG. 14 b . The black curve is the average of 226 normal profiles in thevessel segment. Intensities at non-integer locations are linearlyinterpolated. The red star is the average vessel width across the wholevessel segment marked by observers. The green star is the average vesselwidth across the whole vessel segment measured by proposed method. Thetwo boundaries are flipped and shown in one figure.

The methods provided have high computational performance. For a retinalimage of size 2160×1440 pixels, vesselness map creation takes about 110seconds, and this is an operation

(n), where n is the number of pixels. The image skeletonization andsmall region removal takes about 120 seconds, and this is also anoperation

(n). The total number of centerline pixels is around 20000 pixels (or0.0064n) and the total number of vessel segments is 160. Consequentlythe average length of the graph is around 125 pixels. The average sizeof the graph would be slice number×height of graph×length of graph,which is 45000 (or 0.0145n) in this case. It takes around 9 seconds tobuild all the graphs in the image. The method we used to solve themax-flow problem is a pseudoflow algorithm. The running time is

(n³) [23]. It takes about 41 seconds to solve all the graphs in theexample image.

The method and systems provided for retinal vessel boundary detectionbased on graph search have been validated it on a publicly availabledataset of expert annotated vessel widths. An advantage is in thedetection of both boundaries simultaneously, and is therefore morerobust than methods which detect the boundaries one at a time. Thesimultaneous detection of both borders makes the accurate detectionpossible even if one boundary is of low contrast or blurred. Overall,the method is robust as the only algorithmic parameter is σ in the costfunction, and the final measurement is insensitive to the choice of σ.

One skilled in the art will appreciate that provided is a functionaldescription and that the respective functions can be performed bysoftware, hardware, or a combination of software and hardware. In oneexemplary aspect, the methods and systems can comprise a computer 1501as illustrated in FIG. 15 and described below.

FIG. 15 is a block diagram illustrating an exemplary operatingenvironment for performing the disclosed methods. This exemplaryoperating environment is only an example of an operating environment andis not intended to suggest any limitation as to the scope of use orfunctionality of operating environment architecture. Neither should theoperating environment be interpreted as having any dependency orrequirement relating to any one or combination of components illustratedin the exemplary operating environment.

The present methods and systems can be operational with numerous othergeneral purpose or special purpose computing system environments orconfigurations. Examples of well known computing systems, environments,and/or configurations that can be suitable for use with the systems andmethods comprise, but are not limited to, personal computers, servercomputers, laptop devices, and multiprocessor systems. Additionalexamples comprise set top boxes, programmable consumer electronics,network PCs, minicomputers, mainframe computers, distributed computingenvironments that comprise any of the above systems or devices, and thelike.

The processing of the disclosed methods and systems can be performed bysoftware components. The disclosed systems and methods can be describedin the general context of computer-executable instructions, such asprogram modules, being executed by one or more computers or otherdevices. Generally, program modules comprise computer code, routines,programs, objects, components, data structures, etc. that performparticular tasks or implement particular abstract data types. Thedisclosed methods can also be practiced in grid-based and distributedcomputing environments where tasks are performed by remote processingdevices that are linked through a communications network. In adistributed computing environment, program modules can be located inboth local and remote computer storage media including memory storagedevices.

Further, one skilled in the art will appreciate that the systems andmethods disclosed herein can be implemented via a general-purposecomputing device in the form of a computer 1501. The components of thecomputer 1501 can comprise, but are not limited to, one or moreprocessors or processing units 1503, a system memory 1512, and a systembus 1513 that couples various system components including the processor1503 to the system memory 1512. In the case of multiple processing units1503, the system can utilize parallel computing.

The system bus 1513 represents one or more of several possible types ofbus structures, including a memory bus or memory controller, aperipheral bus, an accelerated graphics port, and a processor or localbus using any of a variety of bus architectures. By way of example, sucharchitectures can comprise an Industry Standard Architecture (ISA) bus,a Micro Channel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, aVideo Electronics Standards Association (VESA) local bus, an AcceleratedGraphics Port (AGP) bus, and a Peripheral Component Interconnects (PCI),a PCI-Express bus, a Personal Computer Memory Card Industry Association(PCMCIA), Universal Serial Bus (USB) and the like. The bus 1513, and allbuses specified in this description can also be implemented over a wiredor wireless network connection and each of the subsystems, including theprocessor 1503, a mass storage device 1504, an operating system 1505,image analysis software 1506, image analysis data 1507, a networkadapter 1508, system memory 1512, an Input/Output Interface 1510, adisplay adapter 1509, a display device 1511, and a human machineinterface 1502, can be contained within one or more remote computingdevices 1514 a, b, c at physically separate locations, connected throughbuses of this form, in effect implementing a fully distributed system.

The computer 1501 typically comprises a variety of computer readablemedia. Exemplary readable media can be any available media that isaccessible by the computer 1501 and comprises, for example and not meantto be limiting, both volatile and non-volatile media, removable andnon-removable media. The system memory 1512 comprises computer readablemedia in the form of volatile memory, such as random access memory(RAM), and/or non-volatile memory, such as read only memory (ROM). Thesystem memory 1512 typically contains data such as image analysis data1507 and/or program modules such as operating system 1505 and imageanalysis software 1506 that are immediately accessible to and/or arepresently operated on by the processing unit 1503.

In another aspect, the computer 1501 can also comprise otherremovable/non-removable, volatile/non-volatile computer storage media.By way of example, FIG. 15 illustrates a mass storage device 1504 whichcan provide non-volatile storage of computer code, computer readableinstructions, data structures, program modules, and other data for thecomputer 1501. For example and not meant to be limiting, a mass storagedevice 1504 can be a hard disk, a removable magnetic disk, a removableoptical disk, magnetic cassettes or other magnetic storage devices,flash memory cards, CD-ROM, digital versatile disks (DVD) or otheroptical storage, random access memories (RAM), read only memories (ROM),electrically erasable programmable read-only memory (EEPROM), and thelike.

Optionally, any number of program modules can be stored on the massstorage device 1504, including by way of example, an operating system1505 and image analysis software 1506. Each of the operating system 1505and image analysis software 1506 (or some combination thereof) cancomprise elements of the programming and the image analysis software1506. Image analysis data 1507 can also be stored on the mass storagedevice 1504. Image analysis data 1507 can be stored in any of one ormore databases known in the art. Examples of such databases comprise,DB2®, Microsoft® Access, Microsoft® SQL Server, Oracle®, mySQL,PostgreSQL, and the like. The databases can be centralized ordistributed across multiple systems.

In another aspect, the user can enter commands and information into thecomputer 1501 via an input device (not shown). Examples of such inputdevices comprise, but are not limited to, a keyboard, pointing device(e.g., a “mouse”), a microphone, a joystick, a scanner, tactile inputdevices such as gloves, and other body coverings, and the like These andother input devices can be connected to the processing unit 1503 via ahuman machine interface 1502 that is coupled to the system bus 1513, butcan be connected by other interface and bus structures, such as aparallel port, game port, an IEEE 1394 Port (also known as a Firewireport), a serial port, or a universal serial bus (USB).

In yet another aspect, a display device 1511 can also be connected tothe system bus 1513 via an interface, such as a display adapter 1509. Itis contemplated that the computer 1501 can have more than one displayadapter 1509 and the computer 1501 can have more than one display device1511. For example, a display device can be a monitor, an LCD (LiquidCrystal Display), or a projector. In addition to the display device1511, other output peripheral devices can comprise components such asspeakers (not shown) and a printer (not shown) which can be connected tothe computer 1501 via Input/Output Interface 1510. Any step and/orresult of the methods can be output in any form to an output device.Such output can be any form of visual representation, including, but notlimited to, textual, graphical, animation, audio, tactile, and the like.

The computer 1501 can operate in a networked environment using logicalconnections to one or more remote computing devices 1514 a, b, c. By wayof example, a remote computing device can be a personal computer,portable computer, a server, a router, a network computer, a peer deviceor other common network node, and so on. Logical connections between thecomputer 1501 and a remote computing device 1514 a, b, c can be made viaa local area network (LAN) and a general wide area network (WAN). Suchnetwork connections can be through a network adapter 1508. A networkadapter 1508 can be implemented in both wired and wireless environments.Such networking environments are conventional and commonplace inoffices, enterprise-wide computer networks, intranets, and the Internet.Any type of network 1515 can be used.

For purposes of illustration, application programs and other executableprogram components such as the operating system 1505 are illustratedherein as discrete blocks, although it is recognized that such programsand components reside at various times in different storage componentsof the computing device 1501, and are executed by the data processor(s)of the computer. An implementation of image analysis software 1506 canbe stored on or transmitted across some form of computer readable media.Any of the disclosed methods can be performed by computer readableinstructions embodied on computer readable media. Computer readablemedia can be any available media that can be accessed by a computer. Byway of example and not meant to be limiting, computer readable media cancomprise “computer storage media” and “communications media.” “Computerstorage media” comprise volatile and non-volatile, removable andnon-removable media implemented in any methods or technology for storageof information such as computer readable instructions, data structures,program modules, or other data. Exemplary computer storage mediacomprises, but is not limited to, RAM, ROM, EEPROM, flash memory orother memory technology, CD-ROM, digital versatile disks (DVD) or otheroptical storage, magnetic cassettes, magnetic tape, magnetic diskstorage or other magnetic storage devices, or any other medium which canbe used to store the desired information and which can be accessed by acomputer.

While the methods and systems have been described in connection withpreferred embodiments and specific examples, it is not intended that thescope be limited to the particular embodiments set forth, as theembodiments herein are intended in all respects to be illustrativerather than restrictive.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; the number or typeof embodiments described in the specification.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which the methods and systems pertain.

It will be apparent to those skilled in the art that variousmodifications and variations can be made without departing from thescope or spirit. Other embodiments will be apparent to those skilled inthe art from consideration of the specification and practice disclosedherein. It is intended that the specification and examples be consideredas exemplary only, with a true scope and spirit being indicated by thefollowing claims.

What is claimed is:
 1. A method for automatic determination ofarteriovenous ratio (AVR) in tissue, comprising: receiving an image;determining a region of interest (ROI) in the image that is centeredaround, but excludes, an optic disc, wherein the ROI comprises aplurality of centerline pixels; extracting, from each of the pluralityof centerline pixels within the ROI, a plurality of color features;normalizing, for each of the plurality of centerline pixels within theROI, the plurality of color features; determining, by a trainedclassifier for each of the normalized plurality of color features foreach of the plurality of centerline pixels within the ROI, a labelindicative of the centerline pixel comprising either an artery or avein; identifying, based on a voting procedure and the label for each ofthe plurality of centerline pixels, arteries and veins in the ROI, theROI having a given diameter, wherein the voting procedure is repeatedfor ROIs at a plurality of different diameters comprising the givendiameter, and wherein the voting procedure votes each centerline pixelas an artery or a vein; determining vessel width measurements for theidentified arteries and veins in the ROI; and estimating, based on thevessel width measurements for the identified arteries and veins in theROI, the AVR.
 2. The method of claim 1, wherein identifying arteries andveins in the region of interest comprises performing vessel segmentationon the image.
 3. The method of claim 2, wherein performing vesselsegmentation on the image further comprises utilizing the trainedclassifier to classify vessel pixels or vessel segments in the image. 4.The method of claim 3, wherein the plurality of color features comprisesone or more derivatives, textures, or color properties.
 5. The method ofclaim 3, wherein vessel tree analysis is used to classify vessel pixelsor vessel segments.
 6. The method of claim 3, wherein blood flow is usedto classify vessel pixels or segments.
 7. The method of claim 1, whereindecreased AVR indicates higher propensity for a disease.
 8. The methodof claim 1, wherein the image is one or more of a color image, amultispectral image, or an Optical Coherence Tomography image.
 9. Themethod of claim 1, wherein the image depicts at least one of: a retina,an iris, skin, a brain surface, or a portion of tissue with visibleblood vessels.
 10. The method of claim 1, wherein determining vesselwidth measurements for the identified arteries and veins comprises usingone or more of a graph search, a multiscale pixel feature basedtobogganing method and splats, or profile fitting.
 11. The method ofclaim 10, wherein the graph search uses a multiscale cost functionderived from a combination of wavelet kernel lifting.
 12. The method ofclaim 1, wherein the ROI is encapsulated by two concentric circleshaving diameters derived from a diameter of the optic disc.
 13. A systemfor automatic determination of arteriovenous ratio (AVR), comprising: amemory; and a processor, coupled to the memory, wherein the processor isconfigured for performing steps comprising: receiving an image;determining a region of interest (ROI) in the image that is centeredaround, but excludes, an optic disc, wherein the ROI comprises aplurality of centerline pixels; extracting, from each of the pluralityof centerline pixels within the ROI, a plurality of color features;normalizing, for each of the plurality of centerline pixels within theROI, the plurality of color features; determining, by a trainedclassifier for each of the normalized plurality of color features foreach of the plurality of centerline pixels within the ROI, a labelindicative of the centerline pixel comprising either an artery or avein; identifying, based on a voting procedure and the label for each ofthe plurality of centerline pixels, arteries and veins in the ROI, theROI having a given diameter, wherein the voting procedure is repeatedfor ROIs at a plurality of different diameters comprising the givendiameter, and wherein the voting procedure votes each centerline pixelas an artery or a vein; determining vessel width measurements for theidentified arteries and veins in the ROI; and estimating, based on thevessel width measurements for the identified arteries and veins in theROI, the AVR.
 14. The system of claim 13, further comprising utilizingthe trained classifier to classify pixels in the image as artery pixelsor vein pixels.
 15. The system of claim 13, wherein decreased AVRindicates higher propensity for a disease.
 16. The system of claim 13,wherein the image depicts at least one of: a retina, an iris, skin, abrain surface, or a portion of tissue with visible blood vessels. 17.The system of claim 13, wherein determining vessel width measurementsfor the identified arteries and veins comprises using one or more of agraph search, a multiscale pixel feature based tobogganing method andsplats.
 18. The system of claim 17, wherein the graph search uses amultiscale cost function, and wherein the multiscale cost function isderived from some combination of wavelet kernel lifting including Gabor,Gaussian derivative, and Difference of Gaussians kernels.
 19. The systemof claim 13, wherein the ROI is encapsulated by two concentric circleshaving diameters derived from a diameter of the optic disc.